Advancing crystal-based fragment screening: investigation of protein crystal environments for improved screening outcomes
(2025) MOBN03 20242Degree Projects in Molecular Biology
- Abstract
- Papain-like protease (PLpro) of SARS-CoV-2 is a vital enzyme involved in viral polyprotein processing and in the suppression of host immune response. Due to its strategic role in viral replication and pathogenesis, PLpro represents a valid yet challenging target for drug development. Fragment-based drug discovery (FBDD) is a powerful strategy employed by the FragMAX facility at MAX IV to identify novel starting points for drug development campaigns, but it requires highly stable and well-diffracting protein crystals to enable the visualization of potential binding fragments through X-ray diffraction. In addition, chemicals necessary for fragment solubility in screening solutions can compromise crystal stability and thus affect the quality... (More)
- Papain-like protease (PLpro) of SARS-CoV-2 is a vital enzyme involved in viral polyprotein processing and in the suppression of host immune response. Due to its strategic role in viral replication and pathogenesis, PLpro represents a valid yet challenging target for drug development. Fragment-based drug discovery (FBDD) is a powerful strategy employed by the FragMAX facility at MAX IV to identify novel starting points for drug development campaigns, but it requires highly stable and well-diffracting protein crystals to enable the visualization of potential binding fragments through X-ray diffraction. In addition, chemicals necessary for fragment solubility in screening solutions can compromise crystal stability and thus affect the quality of results. In this project, we successfully established a high-quality crystal system on which we screened an in-house fragment library. This resulted in the identification of four novel fragments binding to PLpro active site region. These findings may serve as starting point for future FBDD campaigns aimed at discovering inhibitors against PLpro. In parallel, we explored and optimized a glutaraldehyde-based cross-linking method aimed at reinforcing protein crystals when exposed to non-physiological conditions, such as those used in crystallographic fragment-based screens. This approach proved effective in preserving a well-diffracting crystal lattice even under extreme denaturing conditions, highlighting the potential of cross-linking in stabilizing protein crystals. Based on our findings, this method may represent a viable backup strategy that can be implemented in a standard crystallographic fragment screen workflow to ensure successful data collection even when screening conditions compromise crystal integrity. (Less)
- Popular Abstract
- Finding the Coronavirus’ weak spot: how tiny chemical fragments could help block a key viral enzyme
During the COVID-19 pandemic, scientists around the world raced to understand how the virus works and how to stop it. One of its key tools is a protein called PLpro, which the virus uses to make more copies of itself and to weaken our immune defenses. Given the essential role that this protein plays in virus replication, finding a way of blocking its activity could open the way to new antiviral treatments.
In my thesis, I focused on finding small chemical fragments that can stick to PLpro and potentially stop it from working. This approach, known as fragment-based drug discovery, looks for very small molecules that could become the... (More) - Finding the Coronavirus’ weak spot: how tiny chemical fragments could help block a key viral enzyme
During the COVID-19 pandemic, scientists around the world raced to understand how the virus works and how to stop it. One of its key tools is a protein called PLpro, which the virus uses to make more copies of itself and to weaken our immune defenses. Given the essential role that this protein plays in virus replication, finding a way of blocking its activity could open the way to new antiviral treatments.
In my thesis, I focused on finding small chemical fragments that can stick to PLpro and potentially stop it from working. This approach, known as fragment-based drug discovery, looks for very small molecules that could become the starting point for future drugs. To find them, we need high-quality crystals of the protein so we can examine them in fine detail using X-ray light—like taking a microscopic picture.
However, some of the chemicals used in this process can damage the crystals and make it hard to get good results. That’s why I also worked on a method using glutaraldehyde, a type of cross-linking chemical, to make the protein crystals tougher and more stable.
In the end, we discovered four new fragments that bind to the active site of PLpro - which act as a regulator of protein function - and could serve as starting point for future drug development.
Furthermore, we demonstrated that cross-linking could protect the crystals even under harsh conditions, increasing the efficiency and reliability of the whole experimental workflow.
This work helps lay the groundwork for developing new medicines and for making sure the tools we use to discover them are as strong and accurate as possible.
Master’s Degree Project in Molecular Biology: Molecular Genetics and Biotechnology
Credits 60
Department of Biology, Lund University
Advisor: Tobias Krojer
FragMAX facility, MAX IV Laboratory, Lund, Sweden (SE) (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9215044
- author
- Perego, Filippo
- supervisor
- organization
- course
- MOBN03 20242
- year
- 2025
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9215044
- date added to LUP
- 2025-11-07 09:37:40
- date last changed
- 2025-11-07 09:37:40
@misc{9215044,
abstract = {{Papain-like protease (PLpro) of SARS-CoV-2 is a vital enzyme involved in viral polyprotein processing and in the suppression of host immune response. Due to its strategic role in viral replication and pathogenesis, PLpro represents a valid yet challenging target for drug development. Fragment-based drug discovery (FBDD) is a powerful strategy employed by the FragMAX facility at MAX IV to identify novel starting points for drug development campaigns, but it requires highly stable and well-diffracting protein crystals to enable the visualization of potential binding fragments through X-ray diffraction. In addition, chemicals necessary for fragment solubility in screening solutions can compromise crystal stability and thus affect the quality of results. In this project, we successfully established a high-quality crystal system on which we screened an in-house fragment library. This resulted in the identification of four novel fragments binding to PLpro active site region. These findings may serve as starting point for future FBDD campaigns aimed at discovering inhibitors against PLpro. In parallel, we explored and optimized a glutaraldehyde-based cross-linking method aimed at reinforcing protein crystals when exposed to non-physiological conditions, such as those used in crystallographic fragment-based screens. This approach proved effective in preserving a well-diffracting crystal lattice even under extreme denaturing conditions, highlighting the potential of cross-linking in stabilizing protein crystals. Based on our findings, this method may represent a viable backup strategy that can be implemented in a standard crystallographic fragment screen workflow to ensure successful data collection even when screening conditions compromise crystal integrity.}},
author = {{Perego, Filippo}},
language = {{eng}},
note = {{Student Paper}},
title = {{Advancing crystal-based fragment screening: investigation of protein crystal environments for improved screening outcomes}},
year = {{2025}},
}